Cardiac pain is transduced by the activation of cardiac sensory afferents. While the sensation of cardiac pain is primarily caused by myocardial ischemia, the precise chemical mediators are not known. Previous studies indicate protons as a likely mediator of cardiac pain through the sensitization of cardiac afferents via ASIC3, a member of the acid-sensing ion channel family (ASICs). Our preliminary studies show the proton-gated current through ASIC3 and cardiac afferents is enhanced by lactate, which co-localizes with protons during myocardial ischemia. The mechanism is based on a decrease in the concentration of extracellular Ca2+ and Mg2+, suggesting an unexpected role for divalents in ASIC gating. The first aim of this proposal is to determine the mechanism of proton activation of ASICs using electrophysiological and pharmacological techniques. The hypothesis is the binding of protons is not the sole event that causes channel opening. Rather, gating occurs when calcium unbinds from a high-affinity site and protons catalyze this event as they do in any multi-dentate, titratable calcium-binding site (e.g., EGTA). The second aim is to determine which chemicals released by ischemic myocytes are sensed by cardiac afferents. Because acid is but one of the many possible chemical mediators of cardiac pain, a co-culture system of beating myocytes and labeled cardiac afferents will be established to ascertain what molecules mediate the sensation of cardiac pain.